Kinases are enzymes that modify other proteins by chemically adding phosphate groups to them (a process called phosphorylation). Phosphorylation of the targeted proteins results in a functional change of their activity but also can modify association with other proteins, trafficking and subcellular localization. It is estimated that up to 30% of all proteins can be modified by kinases. For this reason kinases are key regulators of majority of cellular pathways, especially those involved in signal transduction. Kinases are currently one of the most interesting and most extensively investigated drug targets. Among the new kinase targets for therapeutic inhibition pursued currently, PIM kinases are one of the most interesting emerging molecular targets. The PIM family of serine-threonine kinases is composed of three highly homologous proteins PIM-1, -2 and -3 which play an important role in intracellular signaling and contribute to pathways involved in cell survival, inflammation, cell movement and stress response (recent reviews please refer to Blanco-Aparicio Biochem Pharmacol. 2012 Oct. 5, Nawijn, Nat Rev Cancer. 2011 January; 11(1):23-34).
With regard to molecular mechanisms of PIM-1 involvement in oncogenic transformation and cancer development, one can point out several processes that are regulated by the PIM-1 kinase like stimulation of cell cycle progression, coactivation of mTOR pathway, inhibition of apoptosis, transcriptional coactivation of c-Myc, promotion of drug resistance and cell migration and metastasis. PIM kinases overexpression has been reported in a variety of cancer types, ranging from hematopoietic malignancies such as diffuse B cell lymphoma, chronic lymphocytic leukemia and acute myelogenous leukemia to solid tumors such as prostate and pancreatic cancer. Acquisition of mutations in the PIM-1 gene can be one of the molecular mechanisms involved in histological transformation of follicular lymphoma (FL) and B-chronic lymphocytic leukemia (B-CLL) to diffuse large B-cell lymphoma (DLBCL) (Rossi et al., Heamatologica, 2006, vol 91, no 10, pp 1405-9). Mutations of the PIM-1 gene have also been detected in cases of AIDS-associated non-Hodgkin lymphoma (Gaidano et al., Blood, 2003, vol 102, no 5, pp 1833-1841), HCV-infected B-cell NHL patients (Libra et al., J. Pathology, 2005, vol 206, Iss 1, pp 87-91), primary central nervous system lymphomas (PCNSLs) (Montesinos-Rongen et al., Blood, Mar. 1, 2004 vol. 103 no. 5 1869-1875), extranodal DLBCL cases and primary cutaneous marginal zone B-cell lymphoma (PCMZL) (Deutsch et al., J Invest Dermatol. 2009 February; 129(2):476-9; Deutsch et al., Blood Apr. 15, 2007 vol. 109 no. 8 3500-3504), primary mediastinal large B-cell lymphoma (PMLBCL) (Martelli et al., Crit Rev Oncol Hematol. 2008 December; 68(3):256-63.). PIM-1 kinase is upregulated in Epstein Barr virus infected B-cells where it enhances transcriptional activity of EBNA2 protein, essential for the growth transformation and immortalization of infected B-cells. This mechanism of action of PIM-1 kinases may predispose immortalized B-cell to undergo malignant transformation (Rainio et al., Virology. 2005 Mar. 15; 333(2):201-6.).
PIM-1 seems to play also a crucial role in development of acute myeloid leukemias (AML). Several reports pointed out a role of PIM-1 kinase in downstream signaling by FLT3 (Fms-like tyrosine kinase 3) kinase. Constitutively activating internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 play an important role in leukemogenesis, and their presence is associated with poor prognosis in AML. Constitutive FLT3 signaling upregulates PIM-1 levels in leukemia cells and the juxtamembrane domain of FLT3 is a critical domain required for this upregulation (Kim et al., Blood. 2005 Feb. 15; 105(4):1759-67; Vu et al., Biochem Biophys Res Commun. 2009 Jun. 5; 383(3):308-13). Interestingly, this downstream signaling seems to be independent of STATS, Akt and MAPK signaling. Up-regulation of PIM-1 kinase contributes to the proliferative and antiapoptotic pathways induced by FLT3 signaling, and the major antiapoptotic mechanism of action is PIM-1 dependent Bad phosphorylation (Kim et al., Br J Haematol. 2006 September; 134(5):500-9). Similarly to FLT3, PIM-1 kinase is also upregulated by the Bcr-Abl fusion protein, a major cause of the chronic myelogenous leukemia. A SH3/SH2 mediated interaction of Bcr/Abl kinase with Hck kinase (hematopoietic cell kinase) lead to activation of Hck and phosphorylation of STAT5B on the critical Tyr699 residue. Activated STAT5B stimulates expression of downstream effectors like PIM-1 kinase and the Al protein, key factors essential for in vitro transformation and in vivo leukemogenesis mediated by Bcr/Abl. (Klejman et al., EMBO J. 2002 Nov. 1; 21(21):5766-74; Nieborowska-Skorska et al., Blood. 2002 Jun. 15; 99(12):4531-9). Whereas inhibition of PIM-1 seems not to be sufficient to overcome Bcr/Abl mediated transformation in cancer cells, an elegant study by Adam et al., showed that PIM-1 and PIM-2 play here redundant roles and simultaneous targeting of the two kinases may be an exciting therapeutic alternative to overcome resistance against small-molecule tyrosine kinase inhibitors (Nosaka and Kitamura, Exp Hematol. 2002 July; 30(7):697-702; Adam et al., Cancer Res. 2006 Apr. 1; 66(7):3828-35.). Involvement of PIM-1 kinase in development of prostate cancer has been extensively studied over the past years and provided several examples of clinical importance and rationale for therapeutic indication. Already in 2001 in a microarrays screen PIM-1 expression was shown to correlate with clinical outcome of the disease and was suggested to be a better marker than the standard diagnostic test for PSA levels in serum (Dhanasekaran et al., Nature. 2001 Aug. 23; 412(6849):822-6). This was further confirmed in studies performed by other groups (Cibull et al., J Clin Pathol. 2006 March; 59(3):285-8; Xu et al., J Surg Oncol. 2005 Dec. 15; 92(4):326-30; Thompson et al., Lab Invest. 2003 September; 83(9):1301-9; Valdman et al., Prostate. 2004 Sep. 1; 60(4):367-71). Overexpression of PIM-1 in human prostate cancer cells induces genomic instability by subverting the mitotic spindle checkpoint, centrosome amplification, chromosome misaggregation and polyploidy. When the PIM-1 kinase is overexpressed in immortalized, non-tumorigenic human cells, these cells became tumorigenic (Roh et al., PLoS One. 2008 Jul. 2; 3(7):e2572; Roh et al., Cancer Res. 2003 Dec. 1; 63(23):8079-84). A very interesting finding by Zemskova and colleagues support additionally use of PIM-1 kinase inhibitors in prostate cancer treatment. Surprisingly, treatment of prostate cancer cells with docetaxel, a standard of care induces STAT3 phosphorylation and transcriptional upregulation of the PIM-1 gene. Expression of PIM-1 kinase was crucial for survival of these cells after docetaxel treatment, as shown by knock down and inhibitor experiments. This data supports further testing of novel, small molecule kinase inhibitors in combination therapies with patients with docetaxel resistance (Zemskova et al., J Biol Chem. 2008 Jul. 25; 283(30):20635-44). In an extensive study by Beier et al., immunohistochemistry experiment performed on cells compared to non-neoplastic tissue showed overexpression of the PIM-1 protein in 98% (41/42) of invasive head and neck squamous cell carcinomas (HNSCC). This study was repeated using primary tumors and metastasis biopsies showing nearly significant correlation of PIM-1 expression with histological tumor, underlining role of PIM-1 in HNSCC developments (Beier et al., Int J Oncol. 2007 June; 30(6):1381-7).
PIM-2 is a second member of the PIM kinase family. Functionally, it has been noticed that PIM-2 overlaps with the Akt/mTOR pathway, but is regulated independently. Both PIM-2 and Akt1 kinase regulate NFκB-dependent transcription by phosphorylation of the Cot kinase (Kane et al., Mol Cell Biol. 2002 August; 22(16):5962-74; Hammerman et al., Cancer Res. 2004 Nov. 15; 64(22):8341-8). It has been indicated that PIM-2 expression maintains high levels of NF-κB activity and NF-κB activation by PIM-2 is required for its antiapoptotic function. Moreover, the data has suggested that Cot-dependent activation of NFκB can occur via the transcriptional induction of PIM-2 rather than as a direct result of a receptor-initiated kinase cascade. Several reports showed that PIM-2 can to some extent substitute or cooperate with PIM-1 in driving tumorigenesis. As both kinases share some of the targets, like the Bad protein, they act both as prosurvival kinases preventing induction of apoptosis (Yan et al., J Biol Chem. 2003 Nov. 14; 278(46):45358-67; Aho et al., FEBS Lett. 2004 Jul. 30; 571(1-3):43-9). As both PIM-1 and 2 are transcriptionally induced by upstream signaling (like FLT3 or Bcr-Abl signaling), they can cooperate and are essential in neoplastic transformation of B-cells by v-Abl oncogene (Chen et al., Blood. 2008 Feb. 1; 111(3):1677-85). Similarly to PIM-1, coexpression of PIM-2 and c-Myc transgene induces malignant transformation (Allen et al., Oncogene. 1997 Sep. 4; 15(10):1133-41). Also the effect on the cell cycle inhibition for both PIM-1 and PIM-2 seem to synergize in accelerating cell proliferation and cell cycle progression as shown in the literature, although the molecular mechanism of cell cycle regulation are described in detail only for PIM-1 kinase (Dai et al., Prostate. 2005 Nov. 1; 65(3):276-86; Chen et al., Mol Cancer Res. 2005 August; 3(8):443-51) There seem however also to be differences between the two kinases. Whereas recent publications on hypoxia point out its emerging role in solid tumor formation and chemoresistance, no similar reports are known for PIM-2 kinase and this role needs to be explored. On the other hand, in the publication by Tamburini, a special emphasis was put on the role of PIM-2 in phosphorylation of crucial 4EBP1 transcription factor (on serine S65) (Tamburini et al., Blood. 2009 Aug. 20; 114(8):1618-27). As shown in this publication, expression of PIM-1 in clinical samples did not correlate with the above finding, providing a proof for non-overlapping role of PIM-1 and PIM-2 in regulation of 4EBP1 phosphorylation, regulation of protein synthesis and promotion of neoplastic transformation. Similar finding were already reported in by Fox and colleagues, stressing out a crucial role of PIM-2 kinase in controlling translation independently from the Akt/mTOR pathway and pointing towards inhibition of PIM-1 kinase as an attractive option for development of new therapies, especially in acute myelogenous leukemia (Fox et al., Genes Dev. 2003 Aug. 1; 17(15):1841-54).
Similarly to PIM-1, overexpression of PIM-2 has been documented in several human tumors types. One of the distinguishing reports is involvement of PIM-2 in tumorigenesis of hepatocellular carcinoma (HCC) (Gong et al., J Surg Res. 2009 May 1; 153(1):17-22). PIM-2 gene expression and its protein levels were investigated in human liver cancer tissues and HepG2 cells (human hepatocellular liver carcinoma cell line). In both cases the expression of PIM-2 gene and protein was higher than in immortalized liver cell line L02, indicating its role as a tumor biomarker. Further experiments indicated that PIM-2 expression and its kinase activity are IL-3 dependent; however its apoptotic inhibition role is IL-3-independent. It was also found that protection against apoptosis by PIM-2 is glucose-dependent, so liver cells growing in vivo, surrounded by high glucose and growth factors concentration have favorable conditions to express PIM-2, however PIM-2 was unable to prevent apoptosis upon glucose deprivation. So once overexpressed in hepatic cells PIM-2 can be an important factor in tumorigenesis.
PIM-3 is the third member of the PIM kinase family. Similarly to PIM-2 and PIM-1, PIM-3 acts in a prosurvival way preventing apoptosis by phosphorylation of Bad. However, in contrast to PIM-1/2, PIM-3 seems to be less specific to Ser112 residue, preferably phosphorylating Ser136, Ser155 and Ser170 (Macdonald et al., BMC Cell Biol. 2006 Jan. 10; 7:1). PIM-3 was the most effective kinase in phosphorylating Ser136 residue, which seems to be crucial for subsequent phosphorylation steps and interaction with the anti-apoptotic Bcl-XL protein. PIM phosphorylation of Bad was therefore found to promote the 14-3-3 binding and inhibition of Bcl-XL binding. Similarly to PIM-1, PIM-3 seems to be also involved in promoting vessel formation and angiogenesis (Zippo et al., Blood. 2004 Jun. 15; 103(12):4536-44; Zhang et al., J Cell Physiol. 2009 July; 220(1):82-90). Angiogenesis is a physiological process involving the growth of new blood vessels from pre-existing vessels. This feature play significant role in tumorigenesis because angiogenesis usually precede metastasis. Although angiogenesis is a normal process in growth and development it is also a fundamental step in the transition of tumors from a dormant state to a malignant one. It was found that PIM-3 is highly expressed both at mRNA and protein levels in endothelial cells and the protein is co-localized at the cellular lamelliopodia focal kinase (FAK), a kinase involved in cellular adhesion and spreading processes. FAK is typically located at structures known as focal adhesions; these are multi-protein structures that link the extracellular matrix to the cytoplasmic cytoskeleton. It is recruited as a participant in focal adhesion dynamics between cells and has a role in motility and cell survival. FAK have also tyrosine kinase activity and originally identified as a substrate for the oncogene protein. After treatment with cytochalasin D which disrupts actin microfilaments, PIM-3 was dispersed from lamelliopodia suggesting strong interaction of PIM-3 with cytoskeleton. Furthermore knockdown of PIM-3 by siRNA had significant effects on endothelial cells migration, proliferation and formation of sprouts. In light of this finding PIM-3 kinase seems to be a new and promising target for novel inhibitors of angiogenesis.
PIM-3 overexpression has been observed in several human cancers, mainly solid tumors like gastrointestinal, colon or liver cancers where expression of PIM-3 seems to be also a poor prognostic marker, however its role in development of pancreatic adenocarcinoma has been studies in more detail (Popivanova et al., Cancer Sci. 2007 March; 98(3):321-8; Zheng et al., J Cancer Res Clin Oncol. 2008 April; 134(4):481-8). PIM-3 was found to be expressed in malignant lesions of the pancreas but not in normal pancreatic tissue (Li et al., Cancer Res. 2006 Jul. 1; 66(13):6741-7). In line with this finding, PIM-3 mRNA and protein were constitutively expressed in all examined human pancreatic cancer cell lines. Knock down of the PIM-1 mRNA levels resulted in apoptosis of the cells, proving essential role of PIM-3 in inhibition of apoptosis in pancreatic cancer cell lines. Further experiments showed that expression of PIM-3 in pancreatic cell lines is controlled by binding of the Ets-1 protein to the 5′-flanking region of human PIM-3 gene between −249 and −183 bp (Li et al., Cancer Sci. 2009 March; 100(3):396-404). Overexpression of Ets-1 transcription factor was able to stimulate transcription and translation of the PIM-3 kinase. These observations indicate that the transcription factor Ets-1 can induce aberrant PIM-3 expression and subsequently prevent apoptosis in human pancreatic cancer cells. Despite the fact that PIM-3 is a kinase of emerging role in cancer development, presented above results implicate how important and diversified roles PIM-3 may play in tumorigenesis and provide rationale for further development of PIM-3 inhibitors for cancer treatment.
DYRK1A/MNB kinase is a member of the dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family, that catalyses the phosphorylation of serine and threonine residues in its substrates as well as the autophosphorylation on a tyrosine residue in the activation loop (Himpel et al, Biochem J. 2001 Nov. 1; 359(Pt 3):497-505, Kentrup et al, J Biol Chem. 1996 Feb. 16; 271(7):3488-95). DYRK1A plays different roles during development, with an important role in controlling brain growth through neuronal proliferation and neurogenesis (Becker FEBS J. 2011 January; 278(2):222, Tejedor FEBS J. 2011 January; 278(2):223-35). Higher than normal levels of DYRK1A are associated with the pathology of neurodegenerative diseases. Especially the trisomy 21-linked Dyrk1A overexpression have been implicated in some neurobiological alterations of Down syndrome, such as mental retardation (Park Cell Mol Life Sci. 2009 October; 66(20):3235-40). Apart from its role in development, it is being increasingly recognised that overexpression of DYRK1A in the adult may contribute to cognitive deficits and Alzheimer-like neurodegeneration in Down syndrome (Wegiel FEBS J. 2011 January; 278(2):236-45). Enhanced phosphorylation of proteins involved in vesicle transport (dynamin, amphiphysin, synaptojanin) might contribute to synaptic dysregulation observed in DYRK1A-overexpressing mice (Murakami J Biol Chem. 2006 Aug. 18; 281(33):23712-24, Adayev Biochem Biophys Res Commun. 2006 Dec. 29; 351(4):1060-5, Xie PLoS One. 2012; 7(4):e34845). Moreover, overexpression of DYRK1A causes hyperphosphorylation of the microtubule-associated protein tau and subsequent formation of neurofibrillary tangles, one of the main pathological hallmarks of Alzheimer's disease or senile dementia (Wegiel FEBS J. 2011 January; 278(2):236-45). Other substrates of DYRK1A have also been identified as components of protein aggregates that are hallmarks of neurodegenerative diseases, such as amyloid plaques in Alzheimer's disease and Lewy bodies in Parkinson's disease and Lewy Body dementia (Kim J Biol Chem. 2006 Nov. 3; 281(44):33250-7). Dyrk1 phosphorylates the human microtubule-associated protein tau at Thr212 in vitro, a residue that is phosphorylated in fetal tau and hyper-phosphorylated in Alzheimer disease (AD) and tauopathies, including Pick disease (Ferrer Neurobiol Dis. 2005 November; 20(2):392-400). DYRK1A polymorphism was recently demonstrated to alter the risk of developing an alpha-synuclein-associated dementia (Jones Neurodegener Dis. 2012; 10(1-4):229-31). The expression of Dyrk1A is elevated in AD brains, when compared with non-diseased human brains (Ferrer Neurobiol Dis. 2005 November; 20(2):392-400; Kimura Hum Mol Genet. 2007 Jan. 1; 16(1):15-23).